The prothrombinase complex is a key enzyme complex of blood coagulation. It consists of the serine protease factor Xa associated, in the presence of Ca2+, with factor Va (a protein cofactor) on an acidic membrane surface. The fully assembled complex catalyzes activation of prothrombin to thrombin (the central and key regulatory enzyme of blood coagulation) many thousand times faster than factor Xa alone. The proposed research will develop a detailed molecular picture of how prothrombin and the components of the prothrombinase interact and assemble on platelet membranes to achieve this essential rate enhancement. Results obtained as part of a Specialized Center for Research in Thrombosis have led to a new working hypothesis to explain the role of protein-lipid interactions in the prothrombin-prothrombinase complex: interactions of specific acidic lipids with certain sites on prothrombin or meizothrombin (the proteolytic intermediate in thrombin formation) trigger protein conformational changes that sequentially direct the appropriate two peptide bonds in prothrombin to the active site of factor Xa so as to increase the rate of thrombin generation. It is presumed also that membrane-induced conformational changes in factors Va and Xa may contribute further to enhancing the efficiency of prothrombin proteolysis on a membrane as opposed to in solution. To test these ideas, the PI will continue to utilize model membrane technology along with a variety of biophysical (fluorescence and FTR spectroscopy, differential scanning calorimetry, quasi-elastic light scattering) and enzymological approaches to pursue five specific aims: 1) isolate and characterize the physical and enzymological properties of native and mutant meizothrombin; 2) continue to model the kinetics of the prothrombinase reaction in terms of thrombin formation via meizothrombin as a transient intermediate; 3) develop a molecular model for the mechanism of factor Va binding to acidic lipid membranes; 4) investigate the presence of a Ca2+independent, phosphatidylserine-specific binding site(s) on prothrombin and factor Xa; 5) monitor membrane-induced conformational changes in prothrombin domains, meizothrombin, and factors Va and Xa. The results will provide insight into the role of specific acidic lipids not only in regulating the prothrombinase complex of blood coagulation but also other extrinsically bound membrane-associated enzymes, such as protein kinase C and phospholipase A2.